Bottom Line:
On the other hand, D283 cell line did not hydrolyze AMP.It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity.In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

ABSTRACTMedulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

pone-0047468-g006: Substrate specificity.Following confluence Daoy, ONS76 and D283 MB cell lines were incubated with different monophosphonucleosides, as described in Materials and Methods section. Bars represent mean ± SD of 6 independent experiments performed in triplicate. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and was taken to indicate statistical significance.

Mentions:
Similar to AMP hydrolysis, the MB cell lines exhibited the same pattern of hydrolysis for other monophosphonucleosides (CMP, GMP, IMP and UMP) (Figure 6), as expected to ecto-5′NT/CD73 [17]. As it was shown before, Daoy and D283 expressed ALP mRNA, another enzyme that can hydrolyze AMP and other phosphate esters. Daoy and ONS76, which presented higher AMPase activity, revealed very low enzymatic activity in the presence of Glu-6P or β-Gly-P, two susbtrates for ALP (Figure 7A). D283 MB cells also poorly hydrolyzed these substrates. In addition, the potential participation of ALP activity in hydrolysis of AMP was evaluated by the presence of levamisole, a specific inhibitor of this enzyme (Figure 7B). AMPase activity in the MB cell lines was unchanged in the presence of the inhibitor, excluding ALP participation in AMP hydrolysis. We observed prominent PPi hydrolysis by all MB cell lines (Figure 7A). Inorganic pyrophosphatase activity was also detected in the three cell lines (Fig. 7A), being in agreement with the presence of an ecto-pyrophosphatase in all MB cell lines analyzed here.

pone-0047468-g006: Substrate specificity.Following confluence Daoy, ONS76 and D283 MB cell lines were incubated with different monophosphonucleosides, as described in Materials and Methods section. Bars represent mean ± SD of 6 independent experiments performed in triplicate. Specific activities were expressed as nmol Pi/min/mg of protein. Data were compared by Two-Way ANOVA test following by Bonferroni post hoc test. (***) p<0.001 and was taken to indicate statistical significance.

Mentions:
Similar to AMP hydrolysis, the MB cell lines exhibited the same pattern of hydrolysis for other monophosphonucleosides (CMP, GMP, IMP and UMP) (Figure 6), as expected to ecto-5′NT/CD73 [17]. As it was shown before, Daoy and D283 expressed ALP mRNA, another enzyme that can hydrolyze AMP and other phosphate esters. Daoy and ONS76, which presented higher AMPase activity, revealed very low enzymatic activity in the presence of Glu-6P or β-Gly-P, two susbtrates for ALP (Figure 7A). D283 MB cells also poorly hydrolyzed these substrates. In addition, the potential participation of ALP activity in hydrolysis of AMP was evaluated by the presence of levamisole, a specific inhibitor of this enzyme (Figure 7B). AMPase activity in the MB cell lines was unchanged in the presence of the inhibitor, excluding ALP participation in AMP hydrolysis. We observed prominent PPi hydrolysis by all MB cell lines (Figure 7A). Inorganic pyrophosphatase activity was also detected in the three cell lines (Fig. 7A), being in agreement with the presence of an ecto-pyrophosphatase in all MB cell lines analyzed here.

Bottom Line:
On the other hand, D283 cell line did not hydrolyze AMP.It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity.In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

ABSTRACTMedulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.